| Early diagnosis of cancer has been considered as a key to control cancer progression,and it has been found that multiple biomarkers including miRNA can work as warning signs of cancer in clinical diagnosis.Fluorescence analysis is a typical method for miRNA detection with the advantages of simple operation and fast signal generation and so on,and it has successfully occupied a prominent position in analytical chemistry field.With decades of development,fluorescence analysis has been commonly combined with a variety of fluorescence amplification strategies for the detection of many biological molecules,thus promoting its application in fields like medical science and chemistry.However,the majority of conventional fluorescence analysis methods are performed in solution,which require huge amounts of samples and long reaction time,unfortunately limiting its implementing to the analysis of trace samples in clinical medicine.Therefore,this thesis designed a new miRNA detection strategy based on the combination of the advantages of fluorescence analysis and photonic crystal(PCs)chips,which not only significantly reduced the requirement amount of sample,but also achieved great amplification of output signal,thus providing useful tools for the accurate detection of tumor related miRNA.The main content of this thesis are as follows:(1)Bioinspired sensor chip for detection of miRNA-21 based on photonic crystals assisted cyclic enzymatic amplification method.PCs with different photonic band gaps(PBGs)were successfully prepared to explore the selective fluorescence enhancement effects of PCs,then the optimized PCs were selected as the platform of chip detection.A biocompatible polydopamine nanomaterial(PDANs)was synthesized.Based on the "off-on" signal change brought by quenching effect of PDANs to fluorescent molecules and the fluorescence recovery in the presence of target molecules,a simple FRET platform was constructed for the preliminary detection of miRNA.Simultaneously,DNase I was added to the FRET system thus forming cyclic enzymatic amplification method(CEAM),which greatly increased the utilization of target molecules and improved the sensitivity of this method.Benefiting from dual amplification effect of PCs and CEAM process,this method only requires 2 ?L sample to achieve the high sensitivity detection of miRNA-21 with a detection limit as low as 50 f M and a linear range of 1 p M – 10 n M.In addition,this method is also suitable for the detection of low abundance of miRNA-21 in biological samples and expected to play an important role in early diagnosis,evaluation and prognosis of cancer.(2)Coupling of photonic crystal and metal-enhanced fluorescence technology for “zero background” detection of miRNA-21In view of the fact that PBG of the prepared PCs partially overlaps fluorescence emission of the employed quantum dots(QDs),the PCs can work as a portable signal amplifier to strengthen the output signal.Furthermore,55 nm silver nanoparticles(Ag NPs)were synthesized to enhance the fluorescence intensity based on the theory of metal enhancement fluorescence(MEF).QDs fluorescent probes and magnetic nanoparticles(MNs)capture probes were prepared to construct a classic sandwich model for miRNA detection.With DNase I treatment and two-step magnetic separation,the background signal interference generated by MNs and QDs can be greatly reduced.The perfect combination of PCs and MEF technology assisting with background reduction method is probably to improve the sensitivity of this method,as well as promote the development and application of a portable miRNA detection device. |
PDF Full Text Request